Aldehyde dehydrogenase and ATP binding cassette transporter G2 (ABCG2) functional assays isolate different populations of prostate stem cells where ABCG2 function selects for cells with increased stem cell activity

Abstract

IntroductionHigh expression of aldehyde dehydrogenase1A1 (ALDH1A1) is observed in many organs and tumors and may identify benign and cancer stem cell populations.MethodsIn the current study, the stem cell characteristics were determined in cells isolated from human prostate cell lines and clinical prostate specimens based upon the ALDEFLUOR™ assay. Cells isolated based on the ALDEFLUOR™ assay were compared to cells isolated based on ATP binding cassette transporter G2 (ABCG2) activity using the side population assay. To test for stem cell characteristics of self-renewal and multipotency, cells with high and low ALDH1A1 activity, based on the ALDEFLUOR™ assay (ALDHHi and ALDHLow), were isolated from prostate clinical specimens and were recombined with rat urogenital sinus mesenchyme to induce prostate gland formation.ResultsThe percentage of ALDHHi cells in prostate cell lines (RWPE-1, RWPE-2, CWR-R1, and DU-145) was 0.5 to 6%, similarly in non-tumor and tumor clinical specimens the percentage of ALDHHi cells was 0.6 to 4%. Recombinants using ALDHHi cells serially generated prostate tissue up to three generations with as few as 250 starting cells. Immunohistochemical analysis of the recombinants using ALDHHi cells contained prostatic glands frequently expressing androgen receptor (AR), p63, chromogranin A, ALDH1A1, ABCG2, and prostate specific antigen (PSA), compared to their ALDHLow counterparts. Inhibition of ALDH resulted in the reduction of sphere formation capabilities in the CWR-R1, but not in the RWPE-2 and DU-145, prostate cell lines. ABCG2 inhibition resulted in a more robust decrease of sphere formation in androgen sensitive cell lines, CWR-R1 and RWPE-2, but not androgen insensitive DU-145. ALDH1A1 expression was enriched in ALDHHi cells and non-side population cells. ABCG2 expression was only enriched in side population cells.ConclusionsThe percentage of ALDHHi cells in prostate cell lines and prostate tissue was consistently higher compared to cells with high ABCG2 activity, identified with the side population assay. The expression of the stem and differentiation markers indicates the ALDHHi recombinants contained cells with self-renewal and multipotency activity. When the two assays were directly compared, cells with the side population phenotype demonstrated more stem cell potential in the tissue recombination assay compared to ALDHHi cells. The increased stem cell potential of side population cells in the tissue recombination assay and the decrease in sphere formation when ABCG2 is inhibited indicates that the side population enriches for prostate stem cells.

Metformin and prostate cancer stem cells: a novel therapeutic target

“…ATP-binding cassette (ABC) transporter ABCG2+ prostate CSCs have also been isolated using the side-population method, which allows for the isolation of cells that can efflux Hoechst 33342 dye more efficiently because of elevated ABC transporter expression. 49,50 Stemness markers have also been used to characterize CSC populations in the prostate. Overexpression of Nanog in LNCaP and DU145 human prostate cancer cell lines promoted CSC properties and enhanced the expression of CD133, CD44, ALDH1A1 and ABCG2.…”

Regulation of hypoxia‐inducible factor functions in the nucleus by sphingosine‐1‐phosphate

“…The sphere formation assay was performed as described previously. 20,34 Briefly, 20 000 cells suspended in DMEM F12 medium (Gibco/Thermo Fisher Scientific) were mixed thoroughly with low growth factor containing BD Matrigel (#354234BD; Biosciences, San Jose, CA, USA) for plating (20 000 cells/well) in an ultra-low attachment 24-well plate (#29443-032; VWR, Radnor, PA, USA) in quadruplicate. The plate was maintained in a 5% of CO 2 incubator at 37°C for 7 days to obtain cancer cell spheroids.…”

Prostate Stem Cells and Cancer Stem Cells

“…However, results using several of these markers may vary depending on the specific cell lines analyzed or assays used, raising concerns as to whether they represent authentic markers of ABCG2 ATP-binding cassette (ABC) transporter family member that may be involved in multidrug resistance. Gangavarapu et al 2013;Samant et al 2015;also Patrawala et al 2005 ALDH Aldehyde dehydrogenase (ALDH) enzymatic activity can be readily detected by Aldefluor assay. Burger et al 2009;van den Hoogen et al 2010…”